The highly GABARAP specific rat monoclonal antibody 8H5 visualizes GABARAP in immunofluorescence imaging at endogenous levels

Indra M Simons,Regina Feederle,Jeannine Mohrlüder,Thomas Zobel,Jochen Dobner,Silke Hoffmann,Elisabeth Kremmer,Nicole Bleffert,Dieter Willbold

doi:10.1038/s41598-018-36717-1

Abstract

The determination of unique functions of GABARAP (gamma-aminobutyric acid type A receptor-associated protein), a member of the highly conserved protein family of mammalian autophagy-related 8 protein (mATG8), within diverse cellular processes remains challenging. Because available anti-GABARAP antibodies perform inadequate, especially within various microscopy-based applications, we aimed to develop an antibody that targets GABARAP but not its close orthologs. Following the latest recommendations for antibody validation including fluorescence protein tagging, genetic and orthogonal strategies, we characterized the resulting anti-GABARAP (8H5) antibody during confocal immunofluorescence imaging in-depth. We compared the antibody staining pattern with that obtained for fluorescence protein tagged GABARAP, GABARAPL1 or GABARAPL2 each ectopically expressed in GABARAP knockout cells. Furthermore, we imaged cells expressing all mATG8 family members at endogenous levels and checked GABARAP knockout cells for unspecific staining under fed or macroautophagy-inducing conditions. Finally, we simultaneously stained cells for endogenous GABARAP and the common autophagosomal marker LC3B. Summarized, the presented antibody shows high specificity for GABARAP without cross-reactivity to other mATG8 family members in immunofluorescence imaging making it a valuable tool for the identification of unique GABARAP functions.

Highlights

The determination of unique functions of GABARAP, a member of the highly conserved protein family of mammalian autophagy-related 8 protein, within diverse cellular processes remains challenging
The respective proteins are divided in two subfamilies: the microtubule-associated protein 1 light chain 3 (MAP1LC3) subfamily including LC3A, LC3B, LC3B2, LC3C, and the GABARAP (γ-amino-butyric acid receptor-associated protein) subfamily with GABARAP, GABARAPL1/ GEC1 (GABARAP-like 1/Glandular epithelial cell protein) and GABARAPL2/GATE-16 (GABARAP-like 2/ Golgi-associated ATPase enhancer of 16 kDa) in humans
Thereby, we focused on immunofluorescence (IF) staining, and included genetic (knockout (KO) cell-based), orthogonal, tagged-target protein expression and independent antibody-based strategies as validation pillars

Summary

Introduction

The determination of unique functions of GABARAP (gamma-aminobutyric acid type A receptorassociated protein), a member of the highly conserved protein family of mammalian autophagy-related 8 protein (mATG8), within diverse cellular processes remains challenging. GABARAP was shown to accumulate in the pericentriolar material from which it can be translocated to forming autophagosomes during starvation[17,18] Despite this apparent progress, elucidation of unique roles for individual ATG8 members within a specific process remains to be a challenging task. Available antibodies against diverse ATG8 family members often show cross-reactivity, and are frequently not sufficiently validated in a transparent manner for the user, especially regarding their performance within diverse applications. As long as such information is lacking, every ATG8-targeted antibody result has to be interpreted with caution unless specificity of the antibody in use has clearly been demonstrated for the application and the ATG8 member chosen[15]

Objectives

Methods

Results

Conclusion